Characterising the RNA-binding protein atlas of the mammalian brain uncovers RBM5 misregulation in mouse models of Huntington’s disease

RNA-binding proteins (RBPs) are key players regulating RNA processing and are associated with disorders ranging from cancer to neurodegeneration. Here, we present a proteomics workflow for large-scale identification of RBPs and their RNA-binding regions in the mammalian brain identifying 526 RBPs. Analysing brain tissue from males of the Huntington’s disease (HD) R6/2 mouse model uncovered differential RNA-binding of the alternative splicing regulator RBM5. Combining several omics workflows, we show that RBM5 binds differentially to transcripts enriched in pathways of neurodegeneration in R6/2 brain tissue. We further find these transcripts to undergo changes in splicing and demonstrate that RBM5 directly regulates these changes in human neurons derived from embryonic stem cells. Finally, we reveal that RBM5 interacts differently with several known huntingtin interactors and components of huntingtin aggregates. Collectively, we demonstrate the applicability of our method for capturing RNA interactor dynamics in the contexts of tissue and disease.


March 2021
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Samples-size calculation was not performed. All MS and Western blot analysis was performed in biological quadruplicates using individual brain samples derived from four individual mouse litter-mates. This choice is motivated by our previous experiments with requirements for label-free quantification and this approach was successfully applied in the paper introducing the pCLAP methodology (Mullari et. al, 2017). All RNA sequencing, Rbm5-CLIP and PCR analysis was done in biological triplicates (using individual brain samples derived from individual mouse litter-mates). CLIP analysis on were performed on individually cultured and transfected replicates. The sample sizes were chosen based about what is required to perform statistical tests consistent with previous publications, and are common practice in the field and our own experience (Fossat et. al, 2023). One replicate from each genotype from the brain-pCLAP experiment done for the comparison of HD and WT samples (data presented in figure 3, supplementary table 2) were excluded due to an unforeseen and unfortunate RNA-degradation in the sample. This is a predefined condition for exclusion and could be determined by the extremely low total sample intensity based on experience with previous experiments.

References
All MS and WB analysis were done in biological quadruplicates, each sample represents an individual mouse brain derived from individual littermates. All RNA-seq, Rbm5-CLIP analysis and PCR amplification experiments were similarly performed in biological triplicates, each sample represents an individual mouse brain from individual littermates. CLIP from cells was done on independent cell culture triplicates. All the replicates were successful (excluding the 2 MS replicates mentioned under data exclusion) and all the data is shown in the manuscript.
Samples were not divided into experimental groups, all replicates for all individual experiments were simultaneously prepared, handled, and statistically processed while taking multiple-hypotheses testing into account.
All samples relating to each experiment were handled simultaneously. During handling, all samples were numbered and processed in random order to avoid introduction of bias into the samples. During MS data acquisition, samples were clearly labeled (and thus not blinded), which is important to MS experimental design. All data analysis was performed with unbiased software, in an unsupervised manner, and therefore blinding is not applicable in this context. All MS data is publicly available and may be re-processed and investigated by any external party.
The following primary antibodies were used for immunoblot analysis in this study, and diluted at 1:1,000. For western-blotting, CLIP and GDP-PD of GFP-tagged candidate RBPs the anti-GFP mouse antibody (11814460001; Roche) was used. For Western-blotting, Rbm5-CLIP analysis and RBM5-CoIP the rabbit polyclonal anti-RBM5 antibody (HPA018011; Merck/Sigma) and mouse monoclonal anti-LUCA15 (200 !g/mL, G-2, sc-515419, Santa Cruz) were used. The Rabbit polyclonal to GAPDH (ab9485) antibody was used as a